WO2006081543A2 - Phosphatase cristalline et procede d'utilisation de celle-ci - Google Patents

Phosphatase cristalline et procede d'utilisation de celle-ci Download PDF

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WO2006081543A2
WO2006081543A2 PCT/US2006/003173 US2006003173W WO2006081543A2 WO 2006081543 A2 WO2006081543 A2 WO 2006081543A2 US 2006003173 W US2006003173 W US 2006003173W WO 2006081543 A2 WO2006081543 A2 WO 2006081543A2
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atom
cdcl4a
polypeptide
phe
tyr
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WO2006081543A3 (fr
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Peter Benjamin Rupert
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Ceptyr, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • This disclosure is directed to a crystalline form of a human protein tyrosine phosphatase designated cdcl4A and more particularly to a crystal of human cdcl4A, a method of crystallization thereof, and its structure, obtained by x-ray diffraction .
  • the disclosure relates to methods of identifying new PTP binding agents and more particularly cdcl4 (A or B) substrates and inhibitors .
  • Chemotherapeutic drugs are a mainstay of cancer treatment . Most of these agents target key steps in DNA replication or cell division. Although chemotherapy targets rapidly growing tumor cells , it can also damage healthy proliferating tissues such as epithelium and bone marrow . Side effects can include hair loss , nausea and vomiting, diarrhea, anemia, and potentially fatal infections caused by neutropenia
  • Reversible protein tyrosine phosphorylation is a key mechanism in regulating many cellular activities . It is becoming apparent that the diversity and complexity of the PTPs and PTKs are comparable , and that PTPs are equally important in delivering both positive and negative signals for proper function of cellular machinery .
  • Regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, proliferation and differentiation . Defects and/or malfunctions in these pathways may underlie certain disease conditions for which effective means for intervention remain elusive including, for example , malignancy, autoimmune disorders , diabetes , obesity, inflammation and infection .
  • the PTP family of enzymes consists of approximately 100 structurally diverse proteins that have in common a highly conserved amino acid PTP catalytic domain, but which display considerable variation in their non-catalytic segments . This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members , which in certain cases have been demonstrated to have specific functions in growth, development and differentiation ( Desai et al . , Cell 84 : 599-609, 1996; Kishihara et al . , Cell 74 : 143-156, 1993; Perkins et al . , Cell 70 : 225-236, 1992 ; Pingel and Thomas , Cell 58 : 1055-1065, 1989; Schultz et al .
  • PTPs participate in a variety of physiologic functions , providing a number of opportunities for therapeutic intervention in physiologic processes through alteration or modulation ( e . g. , up- regulation or down-regulation) of PTP activity.
  • alteration or modulation e . g. , up- regulation or down-regulation
  • therapeutic inhibition of PTPs such as PTPlB in the insulin- signaling pathway may serve to augment insulin action, thereby ameliorating the state of insulin resistance common in Type II diabetes patients .
  • Mitogen-activated protein kinases are components of conserved cellular signal transduction pathways that have a variety of conserved members and that are integral to the cell' s response to stimuli such as growth factors, hormones , cytokines , and environmental stresses .
  • MAP-kinases are activated by phosphorylation by MAP-kinase kinases at a dual phosphorylation motif that has- the sequence Thr-X-Tyr, in which phosphorylation at the tyrosine and threonine residues is required for activity .
  • Activated MAP-kinases phosphorylate several transduction targets , including effector protein kinases and transcription factors .
  • MAP-kinase phosphatases Inactivation of MAP-kinases is mediated by dephosphorylation at the Thr-X-Tyr site by dual- specificity phosphatases referred to as MAP-kinase phosphatases .
  • MAP-kinase phosphatases In higher eukaryotes, the physiological role of MAP-kinase signaling has been correlated with cellular events such as proliferation, oncogenesis , development, and differentiation . Accordingly, the ability to regulate signal transduction via these pathways can lead to the development of treatments and preventive therapies- for human diseases associated with MAP- kinase signaling, such as cancer .
  • dual-specificity protein tyrosine phosphatases dephosphorylate both phosphotyrosine and phosphor- threonine/serine residues (Walton et al . , Ann . Rev. Biochem. 62 : 101-120 , 1993 ) . More than 50 dual-specificity phosphatases that dephosphorylate and inactivate a MAP-kinase have been identified (Shen et al . , Proc. Natl . Acad. Sci .
  • MKP-I WO 97/00315; Keyse and Emslie, Nature 59 : 644-647 ( 1992 ) )
  • MKP-2 WO97/00315
  • MKP-4 MKP-5 , MKP-7 , Hb5 (WO 97 /06245 )
  • PACl Ward et al . , Nature 367 : 651-654 , 1994 )
  • HVH2 Guan and Butch, J. Biol . Chem. 270 : 7197-7203 , 1995
  • PYSTl Room et al . r EMBO J.
  • dual-specificity phosphatases differ in expression, tissue and subcellular distribution, and specificity for MAP-kinase family members . Expression of certain dual-specific phosphatases are induced by stress or mitogens, but others appear to be expressed constitutively in specific cell types . The regulation of dual- specific phosphatase expression and activity is critical for control of MAP-kinase mediated cellular functions , including cell proliferation, cell differentiation and cell survival . For example, dual-specific phosphatases may function as negative regulators of cell proliferation . It is likely that there are many such dual-specific phosphatases , with varying specificity with regard to cell type or activation .
  • cdcl4p in Saccharomyces cerevisiae is an important cell cycle protein phosphatase that regulates exit from mitosis .
  • cdcl4p promotes mitosis by inducing degradation of Clb2p mitotic cyclin (equivalent to human cyclin-B) , through two mechanisms . The first is by upregulation of the mitotic cyclin dependent kinase inhibitor Siclp, and the second is by targeting Clb2p for degradation by the 26S proteasome via ubiquitination .
  • cdcl4p dephosphorylates the Anaphase Promoting Complex (APC) regulatory subunit Cdhlp, making Clb2p cyclin a better substrate for APC mediated ubiquitination .
  • APC Anaphase Promoting Complex
  • cdcl4p is sequestered in the nucleolus by Netlp, however during early anaphase it is released by activation of a pathway referred to as the Mitotic Exit Network (MEN) .
  • MEN Mitotic Exit Network
  • cdcl4A shares 64% identity with yeast cdcl4.
  • cdcl4A is 85% identical to cdcl4B, with the greatest alignment in the catalytic domain .
  • the two cdcl4 homologs vary in sequence predominantly at the N- and C-termini , and cdcl4A has a functional nuclear export sequence while cdcl4B does not .
  • cdcl4B localizes to the nucleolus throughout the cell cycle while cdcl4A is found at the centrosomes (Mailand et al . , Nature Cell Biology; 4 : 317-322 , 2002; Kaiser et al . , MoI Biol Cell; 13 : 2289-2300 , 2002 ) .
  • a biological function for cdcl4B has not been determined, although both cdcl4A and cdcl4B reportedly bind p53 and dephosphorylate pSer-315 in vitro (Li et al . , J Biol Chem 275 : 2410-2414 , 2002 ) .
  • yeast cdcl4p is a master regulator of the inner centromere protein (INCBNP) -Aurora kinase complex (Slil5-Ipll ) , a complex conserved from yeast through mammalian -cells (Pereira and Schiebel, Science, 302 : 2120-2124 , 2003 ) . As part of this complex, yeast cdcl4p regulates mitotic exit by modulating spindle behavior through Slil5-Ipll .
  • cdcl4A is a fundamentally important target for cell cycle progression and cell survival . Modulating cdcl4A with a selective inhibitor would be anticipated to evoke a conflict signal in the growth fraction of human tumors .
  • a cdcl4A inhibitor might be a novel anti-cancer agent expanding the arsenal of drugs available to better manage cancer, having potential synergies with other anti-mitotics , both classical ( e . g. taxanes) and new ( e . g. proteasome inhibitors ) . The design of such an inhibitor would be assisted by more detailed information on the cdcl4A polypeptide, such as crystallographic information .
  • the disclosure provides a crystalline cdcl4A polypeptide .
  • the disclosure further provides the three-dimensional coordinates of a crystalline cdcl4A polypeptide .
  • the disclosure also provides a crystal formed by cdcl4A that diffracts x-ray radiation to produce a diffraction pattern representing the three-dimensional structure of the cdcl4A.
  • the disclosure further provides a method of crystallizing cdcl4A comprising (a) mixing an aqueous solution comprising cdcl4A with a reservoir solution comprising a precipitant to form a mixed volume; and (b) crystallizing the mixed volume .
  • a method for determining a three- dimensional structure of cdcl4A comprising : (a) obtaining crystalline cdcl4A; (b) irradiating the crystalline cdcl4A to obtain a diffraction pattern characteristic of the crystalline cdcl4A; and (c) transforming the diffraction pattern into a three-dimensional structure of the cdcl4A.
  • the disclosure further provides a machine-readable data storage medium comprising a data storage material encoded with machine-readable data that , when read by an appropriate machine, displays a three-dimensional representation of a crystal of a molecule comprising cdcl4A or fragment or variant thereof .
  • a computer means for producing a three-dimensional representation of a cdcl4A crystal, or a cdcl4A crystal : binding agent complex, or co- crystal is included in the present disclosure.
  • the disclosure provides a method for evaluating the potential of a candidate binding agent to associate with a cdcl4A polypeptide or a fragment thereof .
  • the method includes (a) modeling (or producing a three-dimensional representation of) one or more domains of a cdcl4A polypeptide, defined by a plurality of atomic coordinates of the cdcl4A polypeptide; and (b) modeling (or producing a three-dimensional representation of) the association of a candidate binding agent with said modeled cdcl4A polypeptide .
  • the disclosure further provides a computer program on a computer readable medium comprising instructions to cause a computer to : (a) define a cdcl4A polypeptide or fragment thereof based on a plurality of atomic coordinates of the cdcl4A polypeptide; and (b) model (or producing a three-dimensional representation of) a potential binding agent that interacts with the cdcl4A polypeptide . Further disclosed is a computer with display means for displaying the atomic coordinates .
  • the disclosure provides a method of designing a compound that mimics the 3-dimensional surface shape of cdcl4A polypeptide comprising the steps of : (a) determining the 3-dimensional structure of a cdcl4A polypeptide; and (b) designing a compound that complements the 3-dimensional surface configuration of the cdcl4A polypeptide .
  • the disclosure provides a method for determining at least a portion of a three-dimensional structure of a molecular complex, said complex comprising cdcl4A and said method comprising the steps of : (a) determining the structural coordinates of a crystal of a cdcl4A polypeptide; ⁇ b) calculating phases from the structural coordinates ; (c) calculating an electron density map from the phases obtained in step (b) ; and (d) determining the structure of at least a portion of the complex based on said electron density map .
  • the disclosure also provides a method for evaluating the ability of a chemical entity to associate with cdcl4A or a complex thereof .
  • the method includes (a) employing computational or experimental means to perform a fitting operation between the chemical entity and the cdcl4A or complex thereof, thereby obtaining data related to the association; and (b) analyzing the data obtained in step (a) to determine the characteristics of the association between the chemical entity and the cdcl4A or complex thereof.
  • the disclosure provides a heavy-atom derivative of a crystallized form of cdcl4A.
  • the disclosure provides a method of computationally or experimentally evaluating a chemical entity, or binding agent to obtain information about its association with a domain of cdcl4A using a crystal of cdcl4A having the structural coordinates described in Table 2.
  • the disclosure provides a crystalline complex comprising a cdcl4A polypeptide and a candidate binding agent .
  • the disclosure further provides a method for determining the binding of a candidate binding agent to cdcl4A, comprising (a) introducing the candidate binding agent and a crystalline cdcl4A in an environment such that the agent and crystalline cdcl4A can interact; and (b) analyzing the crystalline cdcl4A to determine whether the candidate binding agent binds thereto .
  • the disclosure provides a method of identifying a candidate binding agent for binding to cdcl4A.
  • the method includes (a) constructing a three-dimensional structure of cdcl4A polypeptide using atomic coordinates shown in Table 2 , (b) performing structure-based design of said candidate binding agent using said atomic coordinates of (a) ; and ( c) identifying a candidate binding agent predicted by said structure based design to bind to cdcl4A polypeptide .
  • FIG. IA shows pile-ups of cdcl4Aisol ( SEQ ID NO : 2 ) , cdcl4Aiso2 ( SEQ ID NO : 4 ) , and cdcl4Aiso3 (SEQ ID NO : 6) amino acid sequences in comparison to a consensus amino acid sequence (SEQ ID NO : 13 ) .
  • FIG. 1 shows pile-ups of cdcl4Aisol ( SEQ ID NO : 2 ) , cdcl4Aiso2 ( SEQ ID NO : 4 ) , and cdcl4Aiso3 (SEQ ID NO : 6) amino acid sequences in comparison to a consensus amino acid sequence (SEQ ID NO : 13 ) .
  • IB shows pile-ups of cdcl4Bisol (SEQ ID N0 : 8 ) , cdcl4Biso2 (SEQ ID NO : 10) , and cdcl4Biso3 (SEQ ID NO : 12 ) amino acid sequences in comparison to a consensus amino acid sequence
  • FIG . 2A shows a catalytic domain and representative amino acid residues of cdcl4 with a C-alpha backbone trace of the active site region .
  • FIG . 2B shows a catalytic domain and representative amino acid residues of cdcl4 with an all-atom representation of the active site region in the same orientation as FIG . 2A.
  • FIG . 2C shows a catalytic domain and representative amino acid residues of cdcl4 with an all-atom representation of the active site region rotated 90 ° with respect to FIG . 2B .
  • FIG . 4A shows the nucleic acid sequence of the CDC14Aisol coding sequence (SEQ ID NO : 1 ) .
  • FIG. 4B shows the nucleic acid sequence of the CDC14Aiso2 coding sequence (SEQ ID NO : 3 ) .
  • FIG . 4C shows the nucleic acid sequence of the CDC14Aiso3 coding sequence (SEQ ID NO : 5 ) .
  • FIG . 4D shows the nucleic acid sequence of the CDC14Bisol coding sequence ( SEQ ID NO : 7 ) .
  • FIG . 4E shows the nucleic acid sequence of the CDC14Biso2 coding sequence (SEQ ID NO : 9) .
  • FIG . 4F shows the nucleic acid sequence of the CDC14Biso3 coding sequence (SEQ ID NO: 11 ) .
  • modulating phosphorylation and correlatives thereof such as “modulate” or “modulating” phosphorylation means increasing or decreasing a molecule' s state of phosphorylation relative to a control or normal state .
  • a PTP binding agent e . g. , an inhibitor or an activator
  • An inhibitor of cdcl4 includes any agent that decreases dephosphorylation of a molecule relative to an untreated control .
  • An activator of cdcl4 includes any agent that increases dephosphorylation of a molecule compared to a control lacking such an agent .
  • ⁇ cdcl4A polypeptide or "cdcl4A” each refer to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO : 2 , or to mutants , fragments , variants ( e . g. , SEQ ID Nos : 4 or 6) and conservative substitutions thereof comprising L- or D- amino acids and includes modified forms thereof, such as glycoproteins .
  • a "cdcl4B polypeptide” or w cdcl4B” is intended to encompass a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO : 8 , or to mutants , fragments , variants ( e . g. , SEQ ID Nos : 10 or 12 ) and conservative substitutions thereof comprising L- or D-amino acids and include modified forms thereof, such as glycoproteins .
  • vx cdcl4 polypeptide and "cdcl4" are each intended to encompass either or both a cdcl4A and/or a cdcl4B polypeptide , as the context requires .
  • binding agent means any compound that is capable of binding to or interacting with a cdcl4 polypeptide' s binding domain (e . g. , active site or catalytic site) or other domain ( e. g. , an allosteric or exosite domain) .
  • a binding agent can bind to or interact with a domain on the cdcl4 polypeptide causing a change in the conformation of the polypeptide thereby rendering the cdcl4 inactive .
  • Such compounds can include polypeptides, peptidomimetics , antibodies , antibody fragments , small chemical molecules , and the like .
  • the binding agent may inhibit activity of cdcl4 polypeptide by acting as a competitive binding agent to a naturally occurring substrate of a cdcl4 polypeptide .
  • a binding agent may also be a natural or modified substrate for cdcl4 polypeptide, such as, for example, p53 ⁇ J Biol Chem. 275 ( 4 ) : 2410-4 , 2000) ; hCdhl ( J Biol Chem. 276 (51) : 48237-42 , 2001 ) ; p27Kipl (MoI Biol Cell . 13 ( 7 ) : 2289-300, 2002 ) ; histone Hl (MoI Biol Cell .
  • a binding agent can be a fragment of a naturally occurring polypeptide or a synthetic polypeptide that is designed (based on the disclosure herein) to interact with a binding cavity of a cdcl4 polypeptide .
  • polypeptide refers to a chain of amino acid residues , regardless of length or posttranslational modification (e . g. , glycosylation or phosphorylation) .
  • a polypeptide refers to a polymer in which the monomers are amino acid residues that are j oined together through amide bonds .
  • the amino acids are alpha-amino acids
  • either the L-optical isomer or the D- optical isomer can be used, the L- isomers being typical .
  • a "substantially pure" polypeptide is a polypeptide that has been separated from components that naturally accompany it .
  • a polypeptide is substantially pure when it is at least 60% , by weight, free from the proteins and naturally-occurring molecules with which it is naturally associated .
  • the preparation is at least 75% , 90% , typically 95% , and most typically at least 99% , by weight, free from the proteins and naturally-occurring molecules with which it is naturally associated.
  • a substantially pure polypeptide may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a desired polypeptide; or by chemically synthesizing the polypeptide . Purity can be measured by any appropriate method ( e . g. , column chromatography, polyacrylamide gel electrophoresis , by HPLC analysis , and the like) .
  • polypeptides of the disclosure are intended to cover naturally occurring proteins , as well as those that are recombinantly or synthetically synthesized.
  • Polypeptide fragments are also encompassed by the disclosure . Fragments have fewer amino acid residues than the polypeptides of SEQ ID NO : 2 or SEQ ID NO : 8 , and therein can have the same or substantially the same amino acid sequence as the naturally occurring polypeptide over the corresponding region .
  • a polypeptide or peptide having substantially the same sequence means that an amino acid sequence is largely, but not entirely, the same , and, for purposes of certain aspects of this invention, retains the three-dimensional structure conformation of the sequence to which it is related.
  • the full-length protein may not need to be crystallized in order to provide the benefit of a three- dimensional representation of the conformation of such domain or region .
  • a fragment containing the cdcl4 region or domain of interest can be used and crystallized or even co-crystallized with a candidate binding agent .
  • the representation of the fragment or domain or region can provide valuable information in the design of candidate binding agents , and in particular in rational drug design .
  • polypeptides of the disclosure include peptides , or full length polypeptides or fragments, that contain substitutions , deletions , or insertions into the protein backbone that would still have approximately 70% , 80% , 90% , 95% or 99% homology to the original polypeptide over the corresponding portion of the molecule .
  • a yet greater degree of departure from homology is allowed if like-amino acids, i . e. , conservative amino acid substitutions , are not considered a change in the sequence .
  • a polypeptide that is substantially related to a naturally occurring protein, but for a conservative variation, is also contemplated to be within the methods of the disclosure .
  • a binding agent can be modeled to interact with a variant cdcl4 polypeptide based upon the structural coordinates described herein .
  • a polypeptide binding agent can be modified to generate variants that interact with a cdcl4 polypeptide .
  • a conservative variation denotes the replacement of an amino acid residue by another, biologically similar residue .
  • conservative substitutions include the changes of : alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate ; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine or phenylalanine; tryptophan to phenylalanine; tyrosine to tryptophan or phenylalanine ; and va
  • Modifications and substitutions are not limited to replacement of amino acids .
  • purposes such as increased stability, solubility, or configuration concerns
  • one skilled in the art will recognize the potential value of introducing, (by deletion, replacement, or addition) other modifications .
  • examples of such other modifications include incorporation of rare amino acids , D-amino acids , glycosylation sites, cytosine for specific disulfide bridge formation, and the like .
  • the use of modified polypeptide binding agents that incorporate non-naturally occurring amino acids for increased stability can be developed for therapeutic administration.
  • the modified polypeptide binding agents can be modeled to interact with the binding domain/catalytic domain of a cdcl4A polypeptide based upon the structural coordinates described herein .
  • the modified peptides can be chemically synthesized, or the isolated gene can be site- directed mutagenized, or a synthetic gene can be synthesized and expressed in bacteria, yeast, baculovirus , tissue culture, and the like, to obtain the modified polypeptide .
  • Variations to a cdcl4A polypeptide wherein the polypeptide still retains its biological activity can be made by any number of means known in the art . For example , variations can be obtained by such methods as error-prone PCR, shuffling, oligonucleotide-directed mutagenesis , assembly PCR, sexual PCR mutagenesis, and the like, as well as any combination of two or more thereof .
  • substantially identical means a polypeptide or nucleic acid exhibiting at least 50% , 85% , 90% , but typically at least 95% identity to a reference amino acid or nucleic acid sequence .
  • Identity is often measured using sequence analysis software (e . g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 ) . Such software matches sequences by assigning degrees of homology to various deletions, substitutions and other modifications .
  • sequence analysis software e . g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 .
  • sequence analysis software e . g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 .
  • sequence analysis software e . g. , Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705
  • identity in the context of two or more nucleic acids or polypeptide sequences , refer to two or more sequences or subsequences that are the same or have
  • sequence comparison For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated . Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters .
  • a comparison window includes reference to a segment of any one of the number of contiguous positions falling in the range of about 20 to about 600 , usually from about 50 to about 200, more usually from about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art . Optimal alignment of sequences for comparison can be conducted, e . g.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences , the parameters M (reward score for a pair of matching residues ; always >0 ) .
  • M return score for a pair of matching residues ; always >0
  • a scoring matrix is used to calculate the cumulative score .
  • Extension of the word hits in each direction are halted when : the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment .
  • W wordlength
  • E expectation
  • the disclosure provides a functional model based upon the crystal structure coordinates of cdcl4A. Accordingly, the structure coordinates of cdcl4A, or portions thereof, as provided by this disclosure are particularly useful to assist in solving the structure of a cdcl4 (A or B) mutant . As discussed above, cdcl4A shares a great deal of homology and identity with cdcl4B . A sequence alignment of the cdcl4A and B polypeptide sequences demonstrates the conserved nature of the two molecules ( See FIG . 1A-1B) .
  • a binding agent of a cdcl4A catalytic binding domain identified by the crystal structure coordinates provided herein may interact with a catalytic binding domain of cdcl4B .
  • the active sites of both cdcl4A and B comprise, (from N- to C-) HCKAGLGRT ( e . g. , SEQ ID NO : 2 from amino acid 277-285 (atom 2205 through atom 2268 as shown in Table 2) ; SEQ ID N0: 8 from 313-321 , respectively) in a central loop (FIG . 2A-2C) comprising a catalytic cysteine residue .
  • Other loop sections surround this central loop of cdcl4A and B as set forth in Table 1 below: TABLE 1
  • the disclosure permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including inhibitory compounds, substrates , and the like, capable of binding to a cdcl4A polypeptide alone, or both to a cdcl4A polypeptide and a cdcl4B polypeptide, in whole or in part .
  • One aspect of the disclosure resides in obtaining crystals of the cdcl4A polypeptide of sufficient quality to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods .
  • the knowledge obtained concerning the three-dimensional structure of cdcl4A can be used to assist in the determination of the three-dimensional structure of other PTP proteins .
  • Candidate binding agents can also be designed by various computer models as described herein . Based on the structural coordinates of the cdcl4A polypeptide ( i . e.
  • cdcl4 the three dimensional protein structure
  • small molecules or polypeptides that mimic the shape or configuration or are capable of interacting with, a catalytic domain of a cdcl4 can be designed and synthesized to modulate cdcl4 biological functions (e. g. , modulate dephosphorylation) .
  • the structural coordinates of cdcl4 may be used to design candidate binding agents that bind to non-catalytic regions of cdcl4. Such agents are sometimes called "allosteric" or "exosite” binding agents .
  • the disclosure provides a method of "rational" drug design .
  • Another approach to rational drug design is based on a lead compound that is discovered using high- throughput screens ; the lead compound can be further modified based on a crystal structure of the binding domains of cdcl4A.
  • Such lead compounds and related variants are resynthesized and can be co-crystallized with cdcl4A.
  • another aspect of the disclosure is to provide material , which is a starting material in the rational design of drugs , which modulate the action of cdcl4A.
  • crystal structure coordinates refers to mathematical coordinates derived from mathematical equations related to the patterns obtained from diffraction of a monochromatic beam of X-rays by the atoms ( scattering centers ) of a polypeptide in crystal form ( e . g. , a cdcl4A polypeptide) .
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal .
  • the electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal .
  • the coordinates of the cdcl4A polypeptide can also be obtained by means of computational analysis .
  • Selenomethionine substitution refers to a method of producing a chemically modified form of a crystal of cdcl4A.
  • the cdcl4A polypeptide can be expressed by bacteria in media that is depleted in methionine and supplemented with selenomethionine .
  • Selenium can be thereby incorporated into the crystal in place of the sulfur of methionine .
  • the location ( s ) of selenium are then determined by X-ray diffraction analysis of the crystal . This information is used to generate the phase information used to construct a three- dimensional structure of the protein .
  • Heavy atom derivatization refers to a method of producing a chemically modified form of a crystal of cdcl4A. For example, a crystal is soaked in a solution containing heavy metal atom salts or organometallic compounds, which can diffuse through the crystal and bind to the surface of a polypeptide . The location (s ) of the bound heavy metal atom (s ) are determined by X- ray diffraction analysis of the soaked crystal . This information is used to generate the phase information used to construct a three-dimensional structure of the polypeptide .
  • the term "unit cell” refers to the simplest volume element that by repeated translation describes the crystal .
  • the term "asymmetric unit” refers to the smallest non-repeating element of the unit cell .
  • space group refers to the combination of symmetry operators that when applied to the asymmetric unit describes the contents of the unit cell .
  • the methods of the disclosure allow the modeling and identification of binding agents that can interact with the catalytic domains of both cdcl4A and cdcl4B, or one catalytic domain ( e. g. , a cdcl4A catalytic domain) but not a catalytic domain of the other polypeptide ( e . g. , a cdcl4B) .
  • binding agents for binding domains of related PTPs that share at least 80% , 90% , 95% , 98% or 99% identity to a cdcl4A polypeptide or its binding domain can be identified by the methods and systems of the disclosure .
  • the catalytic domain of a cdcl4A polypeptide includes residues 277-285 of SEQ ID NO : 2 , and can involve the interaction of additional amino acids such as those identified in Table 1.
  • the coordinates of the atoms associated with the crystal structure of the cdcl4A polypeptide are provided in Table 2. More specifically, the atoms associated with the catalytic domain of cdcl4A extend from atom 2205 through atom 2268 (i . e . , amino acid 277 285 of SEQ ID NO : 2 ) .
  • a "binding domain” includes a site (such as an atom, a functional group of an amino acid residue or a plurality of such atoms and/or groups ) in a cdcl4A binding cavity .
  • the binding domain is a catalytic domain, which may interact with a binding agent (e . g. , a substrate, an inhibitor or an activator) .
  • a binding domain may be allosteric .
  • sites may exhibit attractive or repulsive binding interactions , brought about by charge , steric considerations and the like .
  • One approach enabled by the disclosure is to use the structure coordinates as set forth in Table 2 to design binding agents that bind to a cdcl4A polypeptide .
  • the physical properties of the binding agent can be modified in different ways ( e . g. , to alter solubility) .
  • the disclosure enables the design of binding agents that act as inhibitors or substrates of a PTP polypeptide by binding to the cdcl4A molecule .
  • a cdcl4A polypeptide crystal is contacted with a variety of different binding entities to determine optimal sites for interaction between candidate binding agents (e. g. , inhibitors or substrates ) and a cdcl4A binding domain .
  • an approach made possible and enabled by the disclosure is to screen computationally small molecule databases for putative binding entities that can bind in whole, or in part, to a cdcl4A polypeptide or fragment thereof.
  • the quality of fit of such a binding entity to the binding domain may be judged in a variety of ways , e . g. , by shape complementarity or by estimated interaction energy (Meng et al . , J Comp Chem, 13 : 505-524 , 1992 ) .
  • Candidate binding agents can then be synthesized using conventional methods and tested for cdcl4 binding using conventional methods or using those methods described herein.
  • a cdcl4A polypeptide mutant may be crystallized in association or complex with known binding agents, substrates , or inhibitors .
  • the crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of a wild-type cdcl4 molecule . Potential sites for modification within the cdcl4 molecule may thus be identified. This information provides an additional tool for determining the most efficient binding interactions , for example, increased hydrophobic interactions , between a cdcl4 polypeptide and a candidate binding agent or compound.
  • All of the complexes referred to above may be studied using known X-ray diffraction techniques and may be refined versus 2-3 A resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR (Yale University, 1992 , distributed by Molecular Simulations , Inc . ; see also, Methods in Enzymology, vol . 114 and 115 , H. W . Wyckoff et al . , eds . , Academic Press ( 1985 ) . This information may thus be used to design, synthesize and optimize cdcl4 binding agents ( e. g. , inhibitors or substrates ) .
  • cdcl4 binding agents e. g. , inhibitors or substrates
  • binding agents that bind to or inhibit a cdcl4A polypeptide according to the disclosure generally involves consideration of two factors .
  • the binding agent should be capable of physically and structurally associating with a cdcl4A polypeptide .
  • Non-covalent molecular interactions important in the association of a PTP with a substrate, include hydrogen bonding, van der Waals and electrostatic interactions, and the like .
  • the binding agent should be able to assume a conformation that allows it to associate with a cdcl4A polypeptide . Although certain portions of the binding agent will not directly participate in the association, those portions may still influence the overall conformation of the polypeptide .
  • Such conformational requirements include the overall three-dimensional structure and orientation of the binding agent in relation to all or a portion of the binding domain, e . g. , active site or accessory binding site of a cdcl4A polypeptide , or the spacing between functional groups of a compound comprising several chemical entities that directly interact with cdcl4A.
  • the potential inhibitory or binding effect of a binding agent on cdcl4A may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques as described herein or those known in the art using information provided herein . If the theoretical structure of the candidate or test binding agent has insufficient interaction and association between the binding agent and cdcl4A, synthesis and testing of the binding agent may be obviated. However, if computer modeling indicates a potentially strong interaction, the binding agent may then be synthesized and tested for its ability to bind to cdcl4A. Whether or not the binding agent possesses cdcl4A or cdcl4B inhibitory or modulating characteristics can be determined through routine assays .
  • a candidate or test binding agent of cdcl4A or cdcl4B polypeptide may be computationally evaluated and designed by means of a series of steps in which putative binding agents are screened and selected for their ability to associate with the catalytic domain or other areas of cdcl4.
  • One skilled in the art may use one of several methods to screen candidate binding agents for their ability to associate with a cdcl4 polypeptide .
  • This process may begin by visual inspection of, for example, the catalytic domain on a computer screen based on the cdcl4A coordinates provided in Table 2 using methods and equipment described above and elsewhere herein; or used routinely in the art .
  • a computer model of a selected binding agent may then be positioned in a variety of orientations , or docked, within an individual binding pocket .
  • Docking may be accomplished using software such as , and without limitation, QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields , such as , and without limitation, CHARM and AMBER .
  • Specialized computer programs may also assist in the process of selecting candidate binding agents . These include but are not limited to :
  • GRID (Goodford, P . J. , "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules", Med. Chem. , 28 , pp . 849-857 ( 1985 ) ) . GRID is available from Oxford University, Oxford, UK .
  • MCSS (Miranker, A. and M . Karplus , "Functionality Maps of Binding Sites : A Multiple Copy Simultaneous Search Method. " Proteins : Structure . Function and Genetics, 11 , pp . 29-34 ( 1991 ) ) . MCSS is available from Molecular Simulations, Burlington, Mass .
  • AUTODOCK (Goodsell , D . S . and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealling, Proteins : Structure . Function, and Genetics, 8 , pp . 195-202 ( 1990 ) ) .
  • AUTODOCK is available from Scripps Research Institute, La Jolla, Calif .
  • DOCK (Kuntz, I . D. et al . r "A Geometric Approach to Macromolecule-Ligand Interactions", J. MoI . Biol . , 161 , pp . 269- 288 ( 1982 ) ) . DOCK is available from The University of California, San Francisco, Calif .
  • fragments of candidate binding agents are modeled, those fragments can be altered using computer programs .
  • Useful programs to aid one of skill in the art in connecting the individual fragments include : 1.
  • CAVEAT Bartlett, P . A. et al, "CAVEAT : A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules” . In “Molecular Recognition in Chemical and Biological Problems", Special Pub . , Royal Chem. Soc , 78 , pp . 182-196 ( 1989 ) .
  • CAVEAT is available from the University of California, Berkeley, Calif .
  • candidate binding agents may be designed as a whole or "de novo" using either an empty active site or optionally including some portion ( s ) of a known binding site, using methods , such as and without limitation:
  • LUDI (Bohm, et al . , "The Computer Program LUDI : A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp . 61- 78 ( 1992 ) ) .
  • LUDI is available from Biosym Technologies, San Diego, Calif .
  • LEGEND (Nishibata, Y . and A. Itai, Tetrahedron, 47 , p . 8985 ( 1991 ) ) . LEGEND is available from Molecular Simulations , Burlington, Mass .
  • a candidate or test binding agent or compound designed or selected as cdcl4A candidate binding agent may be further computationally optimized so that in its bound state it would lack repulsive electrostatic interaction with the target binding site .
  • Such non-complementary (e . g. , electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions .
  • the sum of all electrostatic interactions between the binding agent and cdcl4A when the binding agent is bound to cdcl4A should have a neutral or favorable contribution to the enthalpy of binding .
  • Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interaction .
  • Examples of programs designed for such uses by way of non-limiting example include : Gaussian 92 , revision C (M . J. Frisch, Gaussian, Inc . , Pittsburgh, Pa . , 1992 ) ; AMBER, version 4.0 (P . A. Kollman, University of California at San Francisco, 1994 ) ; QUANTA/CHARMM (Molecular Simulations , Inc . , Burlington, Mass . 1994 ) ; and Insight H/Discover (Biosysm Technologies Inc . , San Diego, Calif . , 1994 ) .
  • These programs may be implemented, for example, using a Silicon Graphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify the binding properties .
  • initial substitutions are conservative, e. g. , the replacement group will have approximately the same size , shape , hydrophobicity and charge as the original group .
  • Such substituted binding agents may then be analyzed for efficiency of fit to a cdcl4A binding domain by the same computer methods described, above .
  • Other changes may not be conservative and can be used to test groups of differing sizes, charges, and the like .
  • binding agents or modulatory agents as inhibitors or activators by computer fitting kinetic data using standard equations according to Segel , I . H . , Enzyme Kinetics, J. Wiley &Sons , ( 1975 ) .
  • a test binding agent once modeled and synthesized, it can be soaked and then co-crystallized with a cdcl4A polypeptide .
  • the co-crystallization data comprising atomic coordinates can then be analyzed via computer to generate a 3D image of the test agent interacting or associating with the cdcl4A polypeptide .
  • Analysis of the interaction data can lead to the design of a more productive structure-activity-relationship analysis ( commonly referred to in the art as "SAR") , or medicinal chemistry .
  • SAR structure-activity-relationship analysis
  • An iterative process of co-crystallization, analysis , further SAR can greatly enhance the rate at which rational drug design is performed . Described herein are methods of performing "soaks" to obtain co-crystallization data . Further provided are co-crystals of a complex comprising a cdcl4A polypeptide and a binding agent .
  • the crystal structure data provided herein can be used in the design of new or improved binding agents .
  • the cdcl4A polypeptide coordinates can be directly compared to the coordinates of similar enzymes that have inhibitors or substrate bound thereto to give an approximation of the way these and related inhibitors might bind to cdcl4A.
  • the crystal structure of cdcl4A (disclosed) can be overlaid with the crystal structure of cdcl4B ( see, e . g. , EMBO J. 22 ( 14 ) : 3524-35 , 2003) .
  • binding agents that reproduce interaction characteristics similar to those found between a cdcl4A polypeptide and a co-crystallized binding agent (e . g. , a substrate or inhibitor) .
  • a co-crystallized binding agent e . g. , a substrate or inhibitor.
  • detailed knowledge of the nature of binding site interactions allows for the modification of binding agents to alter or improve solubility, pharmacokinetics , and the like, without affecting binding activity. Such modifications can be made using known techniques .
  • Computer programs are widely available that are capable of carrying out the activities necessary to design binding agents using the crystal structure information provided herein . Examples include, but are not limited to, the computer programs listed below :
  • Catalyst/HYPOTM generates models of compounds and hypotheses to explain variations of activity with the structure of binding candidates
  • various general purpose machines may be used with programs written in accordance with the teachings herein, or it may be more convenient to construct more specialized apparatus to perform the operations .
  • the embodiment will typically be implemented in one or more computer programs executing on programmable systems each comprising at least one processor, at least one data storage system (including volatile and non-volatile memory and/or storage elements) , at least one input device, and at least one output device .
  • the program is executed on the processor to perform the functions described herein .
  • Each such program may be implemented in any desired computer language (including machine, assembly, high level procedural , or obj ect oriented programming languages) to communicate with a computer system.
  • the language may be a compiled or interpreted language .
  • the computer program will typically be stored on a storage media or device ( e . g. , ROM, CD-ROM, or magnetic or optical media) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein .
  • the system may also be considered to be implemented as a computer- readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein .
  • Embodiments of the disclosure include systems (e. g. , internet based systems ) , particularly computer systems which store, display and manipulate the coordinate and sequence information described herein .
  • a computer system 100 is illustrated in block diagram form in Figure 3.
  • a computer system refers to the hardware components , software components , and data storage components used to analyze the coordinates (see , e. g. , Table 2 ) and sequences such as those set forth in SEQ ID Nos : 2 , 4 , 6, 8 , 10 , 12 , and Table 2.
  • the computer system 100 typically includes a processor for processing, e . g. , data and instructions , accessing and manipulating the sequence data and structural coordinates .
  • the processor 105 can be any well-known type of central processing unit, such as , for example, the Pentium IV from Intel Corporation, or a similar processor from other suppliers such as Sun, Motorola, Compaq, AMD or International Business Machines .
  • the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components .
  • the processor 105 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components .
  • a skilled artisan can readily appreciate that any one of the currently available computer systems are suitable .
  • the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (typically implemented as RAM) and one or more internal data storage devices 110 , such as a hard drive and/or other computer readable media having data recorded thereon .
  • the computer system 100 further includes one or more data retrieving means 118 for reading the data stored on the internal data storage means 110.
  • the data retrieving means 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, or means for connecting to external data-retrieving means , such as ethernet, a modem capable of connection to a remote data storage system ( e . g.
  • the internal data storage means 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape , and the like, containing control logic and/or data recorded thereon .
  • the computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving means or device .
  • the computer system 100 includes a display means 120 which is used to display output, such as for example a three- dimensional model of a crystalline cdcl4A polypeptide, or a complex comprising the crystalline cdcl4A polypeptide with a candidate binding agent, to a computer user . It should also be noted that the computer system 100 can be linked to other computer systems 125a-c in a network or wide area network to provide centralized access to the computer system 100.
  • substrates of cdcl4A may include full length tyrosine phosphorylated proteins and polypeptides as well as fragments ( e. g. , portions ) , derivatives or analogs thereof that can be phosphorylated at a tyrosine residue and that may, in certain embodiments, also be able to undergo phosphorylation at a serine or a threonine residue .
  • Such fragments, derivatives and analogs include any naturally occurring or artificially engineered cdcl4A substrate polypeptide that retains at least the biological function of interacting with a cdcl4A as provided herein, for example by forming a complex with a cdcl4A polypeptide .
  • cdcl4A polypeptides may be tested for cdcl4A activity using any suitable assay, e. g. , for p53 and/or .APC activity.
  • cdcl4A binds p53 and dephosphorylate pSer-315 in vitro (Li et al . , J Biol Chem 275 : 2410-2414 , 2002 ) .
  • cdcl4A dephosphorylates the APC regulatory protein Cdhl in vitro and that dephosphorylated APCCdhl has activated ubiquitination of cyclin-Bl (Bembenek and Yu, J Biol Chem, 276 : 48237-48242 , 2001 ) .
  • Such assays may be performed in vitro or within a cell-based assay .
  • 32 P-radiolabeled substrate e. g. , p53 or APC
  • kinase reaction resulting in radiolabeled, activated protein .
  • a cdcl4A polypeptide may then be tested (in the presence and absence of an inhibitor) for the ability to dephosphorylate a p53 or APC by contacting the cdcl4A polypeptide with the p53 or APC under conditions sufficient to promote dephosporylation of p53 or APC .
  • Dephosphorylation of the APC may be detected by measuring ubiquitination of cyclin-Bl or measuring the loss of radioactive phosphate groups by ( 1 ) gel electrophoresis , followed by autoradiography; ( 2 ) the shift in electrophoretic mobility following dephosphorylation; (3 ) the loss of reactivity with an antibody specific for phosphotyrosine or phosphothreonine; or ( 4 ) a phosphoamino acid analysis of the p53 or APC protein . Modulation of a cdcl4A activity can be determined by measuring the dephosphorylation activity in the presence and absence of binding agents identified by the methods of the disclosure .
  • a difference in cdcl4A polypeptide dephosphorylation of a p53 or APC or a phosphorylated substrate (such as a tyrosine-, serine- , and/or threonine phosphorylated peptide) in the presence of a binding agent that is greater or less than the amount of dephosphorylation observed in the presence of a comparable amount of native human cdcl4A is indicative of a binding agent that modulates cdcl4A activity.
  • substrates i . e.
  • binding agents ) identified by the methods of the disclosure can be assayed in a similar manner as described herein for p53 or APC, or by using methods known in the art .
  • Candidate binding agents for use in a method of screening for a modulator of cdcl4A according to the disclosure may be provided as "libraries" or collections of compounds , compositions or molecules . Such molecules typically include compounds known in the art as “small molecules” and having molecular weights less than 10 4 , less than 10 3 and preferably less than 10 2 . For example, candidate binding agents will typically have a molecular weight of 300-1000, typical for small molecule agents .
  • members of a library of test compounds can be administered to a plurality of samples , each containing at least one cdcl4A polypeptide as provided herein, and then assayed for their ability to enhance or inhibit cdcl4A-mediated dephosphorylation of, or binding to, a substrate .
  • Compounds so identified as capable of modulating cdcl4A function e . g. , phosphotyrosine and/or phosphoserine/threonine dephosphorylation
  • are valuable for potential therapeutic and/or diagnostic purposes since they may permit treatment and/or detection of diseases associated with cdc!4A activity .
  • Candidate binding agents further may be provided as members of a combinatorial library, which includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels .
  • various starting compounds may be prepared employing one or more of solid-phase synthesis , recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions .
  • the resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures , such as a synthetic combinatorial library of peptides or other compositions that may include small molecules as provided herein (see e . g. , PCT/US94/08542 , EP 0774464 , U . S . 5 , 798 , 035 , U . S . 5, 789, 172 , U . S . 5, 751 , 629, which are hereby incorporated by reference in their entireties ) .
  • Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures , and tested using cdcl4A according to the present disclosure, by first modeling similar compounds and then testing likely candidates in vitro .
  • binding agents may be identified by combining a candidate binding agent with a cdcl4A polypeptide in vitro or in vivo, and evaluating the effect of the candidate binding agent on the cdcl4A phosphatase activity using, for example, a representative assay described herein .
  • An increase or decrease in phosphatase activity can be measured by performing a representative assay provided herein in the presence and absence of a candidate binding agent .
  • a candidate binding agent is modeled using the structural coordinates of a catalytic domain of cdcl4A.
  • Likely candidate binding agents that interact in silico may be included in a mixture of active cdcl4A polypeptide and substrate (e .
  • Cdcl4A activity may also be measured in whole cells transfected with a reporter gene whose expression is dependent upon the activation of an appropriate substrate .
  • appropriate cells i . e. , cells that express cdcl4A
  • reporter gene which may be readily detected using methods well known to those of ordinary skill in the art
  • Dephosphorylation of substrate may be detected based on a decrease in reporter activity .
  • Candidate modulating agents may be added to such a system, as described above , to evaluate their effect on cdcl4A activity .
  • Crystals were grown by the hanging-drop vapor-diffusion method.
  • Purified cdcl4A ( 10-339aa of SEQ ID NO : 2 ) protein was concentrated to ⁇ 10mg/ml ( 0.33 mM) in a buffer containing 700 mM NaCl, 20 mM Tris ( 7.2 ) , 1 mM EDTA, and 2 mM DTT, and stored at -80 0 C .
  • Drops consisting of 2 ⁇ l of protein solution were mixed with 2 ⁇ l of reservoir solution (200- 250 mM MgCl 2 , 50 mM Tris ( 8.0 ) , 16-18%PEG 8000 , 5 mM DTT) on a coverslip.
  • the coverslip was inverted and allowed to equilibrate over a 0.5 ml reservoir at 23 0 C .
  • a greased seal isolated the hanging drop and the reservoir solution from the exterior environment . Over the course of several days the hanging drop equilibrated to the same osmotic strength as the reservoir and small protein crystals appeared ( 80 x 80 x 400 mm 3 ) .
  • the crystals were identified and selected for exposure to X-rays , they were transferred first to a cryo-solution and then flash- cooled .
  • the cryo-solution is identical to the reservoir solution except that the PEG 8000 is increased -5% and 10% 2-methy1-2 , 4- pentanediol (MPD) is included to impede ice formation .
  • MPD 4- pentanediol
  • the active site is comprised of a central loop, amino acid residues 277 to 285 (atom 2205 through atom 2268 as shown in Table 2 ) , which contains the catalytic cysteine 278 , as well as several additional loop sections that surround the central loop and provide residues for making specific contacts to substrates and inhibitors .
  • the additional loop sections are comprised of residues 46 to 49, 131 to 135 , 173 to 181 , 191 to 195 , 204 to 206, 227 to 229, 249 to 253, and 312 to 320 ( see, e . g. , FIG. 2A- 2C) .
  • Methods for soaking candidate binding agents into the active site Crystals were stabilized in a solution comprising approximately 50 mM Tris ( 8.0 ) , 25 mM MgCl 2 , 50 mM NaCl, 5 mM DTT, 10% MPD, and 20% PEG8000 overnight . These crystals were then transferred to an identical solution that contains about 4- 10 mM of candidate binding agent . After equilibrating for several hours in the candidate binding agent-containing solution, crystals were flash-cooled by plunging into liquid nitrogen . X- ray diffraction data was collected on a Raxis IV using 1.54 A wavelength CuKa X-rays generated from a rotating anode .
  • Crystals were mounted in cryoloops and maintained at about 150 K ( -120 0 C) throughout the data collections in a cryo stream. After the diffraction data were collected, the images were processed and reduced to a scaled and indexed set of unique intensities . The protein structural content of the unit cell was then determined by molecular replacement technique .
  • the initial search model is the homologous protein cdcl4B (Pdb accession number : lOHC) . After the correct rotation and translation has been applied to the search model, subsequent rounds of conventional refinement and manual model building are performed . [00108] TABLE 2 is provided below :
  • REMARK CROSS-VALIDATION METHOD THROUGHOUT REMARK FREE R VALUE TEST SET SELECTION : RANDOM REMARK R VALUE (WORKING + TEST SET) : 0.19653 REMARK R VALUE (WORKING SET) : 0.19159 REMARK FREE R VALUE : 0.29120 REMARK FREE R VALUE TEST SET SIZE (%) : 5.0 REMARK FREE R VALUE TEST SET COUNT : 747 REMARK REMARK FIT IN THE HIGHEST RESOLUTION BIN.

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Abstract

L'invention concerne une forme cristalline d'une protéine tyrosine-phosphatase humaine appelée cdcl4A et, plus précisément, un cristal de la cdcl4A humaine, un procédé de cristallisation de celui, la structure de celui-ci obtenue par diffraction de rayons x. De plus, l'invention concerne des procédés d'identification d'agents de liaison PTP et, plus précisément, des substrats et inhibiteurs de cdcl4 (A ou B).
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MAILAND ET AL.: 'Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation.' NATURE CELL BIOLOGY. vol. 4, April 2002, pages 317 - 322 *
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